EPR detection of protein-derived radicals in the reaction of H2O2 with fe bound in mitochondrial F(1)ATPase

A severe inactivation is obtained upon the addition of H2O2 to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H2O2 produces the complete oxidation of Fe(II) to Fe(III) an d the concomitant appearance of two protein-derived radical species. The tw o signals (g = 2.036 and g = 2.007) display a different temperature depende nce and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers, Such events are not observed when H2O2 is added either to native F(1)ATPase con taining a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(II I) and Fe(II), intramolecular long-range electron transfer may occur from F e(II) to a high oxidation state species of Fe formed in the nucleotide-inde pendent site upon oxidation of Fe(III) by H2O2. (C) 1999 Academic Press.